Targeted at learning about inflammatory eye diseases, this project focused in FY 2001 on two topics: (1) pathogenic mechanisms of immune-mediated ocular inflammation and the molecules involved in the process and (2) investigation of the immunopathogenic capacity of RPE65. Topic 1. The experimental system developed in FY 2000, in which ocular inflammation is induced by T-helper cells of type 1 (Th1) or type 2 (Th2), was used to analyze the involvement of cytokines, chemokines and chemokine receptors in each of these diseases. Noteworthy observations include: (a) upregulation of large numbers of molecules of the three families was observed, with three patterns observed, i.e., preferential expression in eyes with inflammation induced by Th1-cells, by Th2 cells, or similar expression in eyes diseased by either cell type; (b) surprisingly, high levels of nine of these inflammation-related molecules were detected in intact normal eyes; (c) the expression pattern of inflamatory molecules in mouse eyes developing experimental autoimmune uveoretinitis (EAU) paralleled that in eyes with Th1-induced disease; (d) both infiltrating leukocytes and retinal pigment epithelium (RPE) cells expressed chemokines in distinct but overlapping patterns in inflamed eyes. This study represents the most comprehensive analysis to date of inflammatory mediator expression during the development of ocular inflammation, as well as of the expression of inflammation-related molecules in Th1- versus Th2-cell-induced disease. Topic 2. RPE65, a protein that plays a pivotal role in the vision process, is expressed specifically in the RPE cell layer. When injected into Lewis rats, RPE65 induced EAU at doses as low as 1 microgram, i.e., a minimal dose similar to that of arrestin, a well known uveitogenic retinal protein. RPE65 induced EAU in three other inbred rat strains. Similar to disease induced by other uveitogenic proteins, EAU induced by RPE65 was found to be cell-mediated, as indicated by the finding that this disease can be adoptively transferred to naive recipients by sensitized lymphocytes. On the other hand, unlike arrestin and several other uveitogenic proteins, immunization with RPE65 did not induce inflammation in the pineal gland, the so-called third eye in vertebrates.